Exportin 1 (XPO1/CRM1) is the principal nuclear export receptor for cargos bearing hydrophobic nuclear export sequences (NESs). Dysregulation of XPO1-dependent export is implicated in cancer, neurodegeneration, and other diseases, yet a comprehensive view of XPO1 function remains limited by the poor reliability of sequence-based NES prediction. Existing predictors are largely derived from a small set of XPO1-cargo structures and are therefore biased toward canonical docking geometries, limiting their ability to detect NESs that engage XPO1 through noncanonical pocket-occupancy patterns. We hypothesized that deep learning-based structural modeling could overcome this limitation by directly sampling binding geometries. Using AlphaFold 3, we modeled full-length cargo-XPO1-RanGTP complexes for more than 4,000 human proteins and identified over 3,000 previously uncharacterized, high-confidence NESs. Integration of AlphaFold predictions with unsupervised structural geometry analysis and experimental validation identified both canonical NESs and noncanonical sequence patterns exhibiting atypical anchor-residue usage, expanding the structural language of XPO1-recognized NESs. Groove-resolved contact maps further revealed helix rotation within the export groove as a regulatory feature that can rewire pocket usage without altering the core NES sequence, enabling PTM- and cofactor-sensitive tuning of export strength. This exportome atlas resolves many previously ambiguous or unidentified NESs in disease-associated proteins and across major cellular systems, including centrosome organization, mRNA processing, ubiquitin signaling, kinase networks, ribosome quality control, and macroautophagy. We further identified recurrent NES-NLS tandem motifs encoded in primary sequence, suggesting coordinated regulation of nucleocytoplasmic transport. Together, our deep learning-based exportome atlas, integrated with NLS maps and accessible through a web-searchable resource, defines an expanded and regulatable code of nuclear transport at proteome scale and offers a framework for dissecting nuclear trafficking and its dysregulation in human disease.

PAS domains are ubiquitous sensory modules that transduce environmental signals into cellular responses through tandem PAS folds and PAS-associated C-terminal (PAC) motifs. While this conserved architecture underpins their regulatory roles, here we uncover a structural divergence in the metazoan PAS domain-regulated kinase (PASK). By integrating evolutionary-scale domain mapping with deep learning-based structural models, we identified two PAS domains in PASK, namely PAS-B and PAS-C, in addition to the previously known PAS-A domain. Unlike canonical PAS domains, the PAS fold and PAC motif in the PAS-C domain are spatially segregated by an unstructured linker, yet a functional PAS module is assembled through intramolecular interactions. We demonstrate that this assembly is nutrient responsive and serves to remodel the quaternary structure of PASK that positions the PAS-A domain near the kinase activation loop. This nutrient-sensitive spatial arrangement stabilizes the activation loop, enabling catalytic activation of PASK. These findings revealed an alternative mode of regulatory control in PAS sensory proteins, where the structural assembly of PAS domains links environmental sensing to enzymatic activity. By demonstrating that PAS domains integrate signals through dynamic structural rearrangements, this study broadens the understanding of their functional and regulatory roles and highlights potential opportunities for targeting PAS domain-mediated pathways in therapeutic applications.

The ligand-regulated PAS domains are one of the most diverse signal-integrating domains found in proteins from prokaryotes to humans. By biochemically connecting cellular processes with their environment, PAS domains facilitate an appropriate cellular response. PAS domain-containing Kinase (PASK) is an evolutionarily conserved protein kinase that plays important signaling roles in mammalian stem cells to establish stem cell fate. We have shown that the nuclear translocation of PASK is stimulated by differentiation signaling cues in muscle stem cells. However, the mechanistic basis of the regulation of PASK nucleo-cytoplasmic translocation remains unknown. Here, we show that the PAS-A domain of PASK contains a putative monopartite nuclear localization sequence (NLS) motif. This NLS is inhibited in cells via intramolecular association with a short linear motif, termed the PAS Interacting Motif (PIM), found upstream of the kinase domain. The interaction between the PAS-A domain and PIM is evolutionarily conserved and serves to retain PASK in the cytosol in the absence of signaling cues. Consistent with that, we show that metabolic inputs induce PASK nuclear import, likely by disrupting the PAS-A: PIM association. We suggest that a route for such linkage may occur through the PAS-A ligand binding cavity. We show that PIM recruitment and artificial ligand binding to the PAS-A domain occur at neighboring locations that could facilitate metabolic control of the PAS-PIM interaction. Thus, the PAS-A domain of PASK integrates metabolic signaling cues for nuclear translocation and could be targeted to control the balance between self-renewal and differentiation in stem cells.

The Per-Arnt-Sim (PAS) domains are characterized by diverse sequences and feature tandemly arranged PAS and PAS-associated C-terminal (PAC) motifs that fold seamlessly to generate the metabolite-sensing PAS domain. Here, using evolutionary scale sequence, domain mapping, and deep learning-based protein structure analysis, we deconstructed the sequence-structure relationship to unearth a novel example of signal-regulated assembly of PAS and PAC subdomains in metazoan PAS domain-regulated kinase (PASK). By comparing protein sequence, domain architecture, and computational protein models between fish, bird, and mammalian PASK orthologs, we propose the existence of previously unrecognized third PAS domain of PASK (PAS-C) formed through long-range intramolecular interactions between the N-terminal PAS fold and the C-terminal PAC fold. We experimentally validated this novel structural design using residue-level cross-linking assays and showed that the PAS-C domain assembly is nutrient-responsive. Furthermore, by combining structural phylogeny approaches with residue-level cross-linking, we revealed that the PAS-C domain assembly links nutrient sensing with quaternary structure reorganization in PASK, stabilizing the kinase catalytic core of PASK. Thus, PAS-C domain assembly likely integrates environmental signals, thereby relaying sensory information for catalytic control of the PASK kinase domain. In conclusion, we theorize that during their horizontal transfer from bacteria to multicellular organisms, PAS domains gained the capacity to integrate environmental signals through dynamic modulation of PAS and PAC motif interaction, adding a new regulatory layer suited for multicellular systems. We propose that metazoan PAS domains are likely to be more dynamic in integrating sensory information than previously considered, and their structural assembly could be targeted by regulatory signals and exploited to develop therapeutic strategies.

Quiescent stem cells are activated in response to a mechanical or chemical injury to their tissue niche. Activated cells rapidly generate a heterogeneous progenitor population that regenerates the damaged tissues. While the transcriptional cadence that generates heterogeneity is known, the metabolic pathways influencing the transcriptional machinery to establish a heterogeneous progenitor population remains unclear. Here, we describe a novel pathway downstream of mitochondrial glutamine metabolism that confers stem cell heterogeneity and establishes differentiation competence by countering post-mitotic self-renewal machinery. We discovered that mitochondrial glutamine metabolism induces CBP/EP300-dependent acetylation of stem cell-specific kinase, PAS domain-containing kinase (PASK), resulting in its release from cytoplasmic granules and subsequent nuclear migration. In the nucleus, PASK catalytically outcompetes mitotic WDR5-anaphase-promoting complex/cyclosome (APC/C) interaction resulting in the loss of post-mitotic Pax7 expression and exit from self-renewal. In concordance with these findings, genetic or pharmacological inhibition of PASK or glutamine metabolism upregulated Pax7 expression, reduced stem cell heterogeneity, and blocked myogenesis in vitro and muscle regeneration in mice. These results explain a mechanism whereby stem cells co-opt the proliferative functions of glutamine metabolism to generate transcriptional heterogeneity and establish differentiation competence by countering the mitotic self-renewal network via nuclear PASK.

Tissue regeneration is a complex molecular and biochemical symphony. Signaling pathways establish the rhythmic proliferation and differentiation cadence of participating cells to repair the damaged tissues and repopulate the tissue-resident stem cells. Sensory proteins form a critical bridge between the environment and cellular response machinery, enabling precise spatiotemporal control of stem cell fate. Of many sensory modules found in proteins from prokaryotes to mammals, Per-Arnt-Sim (PAS) domains are one of the most ancient and found in the most diverse physiological context. In metazoa, PAS domains are found in many transcription factors and ion channels; however, PAS domain-containing Kinase (PASK) is the only metazoan kinase where the PAS sensory domain is connected to a signaling kinase domain. PASK is predominantly expressed in undifferentiated, self-renewing embryonic and adult stem cells, and its expression is rapidly lost upon differentiation, resulting in its nearly complete absence from the adult mammalian tissues. Thus, PASK is expressed within a narrow but critical temporal window when stem cell fate is established. In this review, we discuss the emerging insight into the sensory and signaling functions of PASK as an integrator of metabolic and nutrient signaling information that serves to balance self-renewal and differentiation programs during mammalian tissue regeneration.

The cell cycle offers a unique opportunity for stem cells to sample metabolic and signaling cues to establish cell fate. Molecular pathways that integrate and convey these signals to cell cycle machinery to license cell fate transitions and drive terminal differentiation remain unknown. Here, we describe a signaling role of mitochondrial glutamine metabolism in driving exit from cell cycle-linked self-renewal to generate differentiation competent progenitors. In proliferating stem cells, mitochondrial glutamine metabolism opposes the WDR5-linked self-renewal network via acetylation and nuclear translocation of its upstream regulator, PASK. Nuclear PASK disrupts the mitotic WDR5-anaphase-promoting complex (APC/C) interaction to drive exit from self-renewal. Consistent with these roles, loss of PASK or inhibition of glutamine metabolism preserves stemness in vitro and in vivo during muscle regeneration. Our results suggest a mechanism whereby the proliferative functions of glutamine metabolism are co- opted by stem cells to establish cell fate.

Stem cell fate in the tissue niche is intimately connected with intracellular metabolic state and the extra‐cellular hormonal stimulations. We have shown that a sensory kinase, PAS domain Kinase (PASK) phosphorylates Wdr5, a member of COMPASS family of histone methyltransferases, to activate the stem cell differentiation program in multiple differentiation paradigms, in vivo and in vitro (eLife, 2016). PASK is expressed highly and exclusively in stem cells, yet the differentiation signaling cues are required to activate PASK and its downstream functions, suggesting PASK remains inaccessible to bind Wdr5 in proliferating stem cells. Here, we show that the mechanistic Target of Rapamycin (mTOR) phosphorylates PASK to promote Wdr5 recruitment and myogenesis in response to nutrient and hormonal signaling. How mTOR phosphorylation stimulates PASK‐Wdr5 interactions remains unknown but is a key mechanistic question that could help understand how nutrient and metabolic signaling can acutely control stem cell differentiation on demand. By using multi‐disciplinary approaches, we show that the PAS domain of PASK inhibits catalytic activity of the kinase domain. Interestingly, mTOR stimulated phosphorylation induces a conformational change resulting in the increased Wdr5 binding, and catalytic activity of PASK. Thus, our data show how information pertaining to nutrient availability is communicated to epigenetic complexes via sequential activation of the mTORC1‐PASK‐Wdr5 pathway.Support or Funding InformationNIH R01: Epigenetic Control of muscle stem cell function by PASK‐Wd5 signaling pathway.1R01AR073906‐01A1

To determine how nutrient signaling impacts stem cell functions PASK phosphorylation: We measured in situ phosphorylation of PASK by metabolic 32P labeling of stem cells expressing WT or mutant versions of PASK. PASK Activation: PASK activation was measured using in vitro kinase assay using radio-labeled ATP. Myogenesis: Myogenesis was measured by immunohistological, and immunofluorescent analysis of differentiating muscle stem cells. Antibodies used were: Myogenin (F5D-Developmental Hybridoma), MF20 (Myosin heavy chain), Pax7 and MyoD. Stem cell fate in the tissue niche is intimately connected with intracellular metabolic state and the extracellular hormonal stimulations. We have identified PAS domain containing Kinase (PASK) as a stem cell enriched protein kinase that is required for establishment of the differentiation program in many stem cell paradigms. For this function, PASK phosphorylates Wdr5, a member of the COMPASS family of histone methyltransferases, to activate the epigenetic processes required for the stem cell differentiation (eLife, 2016). Here we show that a master nutrient sensor, mTOR complex 1 (mTORC1) activates PASK via multi-site phosphorylation during stem cell differentiation. This phosphorylation of PASK by mTORC1 is required for epigenetic activation of the Myogenin transcription, exit from the self-renewal and induction of the myogenesis program. Our data suggest that mTORC1-PASK signaling generates MyoG + committed myoblasts (epigenetically - an early stage of myogenesis), whereas mTORC1-S6K1 signaling is required for myoblast fusion (translationally - later stage of myogenesis). Our discoveries show that nutrient signaling can partition stem cell fates during different stages of the myogenesis program downstream of mTOR signaling via activation of two distinct protein kinases. NIH R01 (Chintan Kikani), HHMI (Jared Rutter)

During skeletal muscle regeneration, muscle stem cells (MuSCs) respond to multiple signaling inputs that converge onto mammalian target of rapamycin complex 1 (mTORC1) signaling pathways. mTOR function is essential for establishment of the differentiation-committed progenitors (early stage of differentiation, marked by the induction of myogenin expression), myotube fusion, and, ultimately, hypertrophy (later stage of differentiation). While a major mTORC1 substrate, p70S6K, is required for myotube fusion and hypertrophy, an mTORC1 effector for the induction of myogenin expression remains unclear. Here, we identified Per-Arnt-Sim domain kinase (PASK) as a downstream phosphorylation target of mTORC1 in MuSCs during differentiation. We have recently shown that the PASK phosphorylates Wdr5 to stimulate MuSC differentiation by epigenetically activating the myogenin promoter. We show that phosphorylation of PASK by mTORC1 is required for the activation of myogenin transcription, exit from self-renewal, and induction of the myogenesis program. Our studies reveal that mTORC1-PASK signaling is required for the rise of myogenin-positive committed myoblasts (early stage of myogenesis), whereas mTORC1-S6K signaling is required for myoblast fusion (later stage of myogenesis). Thus, our discoveries allow molecular dissection of mTOR functions during different stages of the myogenesis program driven by two different substrates.

Stem cell fate in the tissue niche is intimately connected with intra- and extra-cellular metabolic state and the hormonal stimulations. We have shown that a sensory kinase, PAS domain Kinase (PASK) phosphorylates Wdr5, a member of COMPASS family of histone methyltransferases, to activate the stem cell differentiation program in multiple paradigms (elife, 2016). PASK is expressed highly and exclusively in stem cells, yet the differentiation signaling cues are required to activate PASK and its downstream functions, suggesting PASK remains inaccessible to bind Wdr5 in proliferating stem cells. Our recent data showed that the mechanistic Target of Rapamycin (mTOR) activates PASK-Wdr5 signaling pathway in response to the differentiation signaling cues such as nutrients and insulin (in peer-review). mTOR induces multi-site phosphorylation on PASK to stimulate the recruitment of Wdr5 by PASK, resulting in Wdr5 phosphorylation at Ser49. Phosphorylation of Wdr5 stimulates the histone trimethylation at lysine 4 (H3K4me3) at the Myogenin promoter to activate the muscle stem cell differentiation program. How mTOR phosphorylation stimulates PASK-Wdr5 interactions remains unknown but is a key mechanistic question that could help understand how nutrient and metabolic signaling could acutely control stem cell differentiation on demand. By using multi-disciplinary approaches, we show that the PAS domain of PASK negatively regulates PASK-Wdr5 interaction by occupying the Wdr5 interacting surface onto PASK. Interestingly, we have mapped mTOR stimulated phosphorylation sites on PASK adjacent to a region where both PAS and Wdr5 binds. We show that mTOR phosphorylation serves to disrupt intra-molecular inhibition of PAS domain, allowing for Wdr5 binding and phosphorylation by PASK. As PAS domains are versatile sensors of the metabolic environment and bind diverse sets of ligands such as heme, oxygen, blue light and organic small molecules, our data also suggest a possibility of additional allosteric regulation of PASK activity via PAS domains which could impact stem cell fate. In proliferating stem cells, PASK expression is high, but it is kept inaccessible to bind Wdr5 by intramolecular association between PAS domain and PAS interacting motif (PIM). PIM is situated adjacent to Wdr5 binding motif and mTOR phosphorylation sites. Upon differentiation signaling, mTOR mediated phosphorylation induces local conformation change allowing PIM to be dissociated from PAS domain resulting in Wdr5 binding onto PASK, its phosphorylation and the onset of differentiation. This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

In the tissue microenvironment, stem cell functions are modulated by extrinsic signaling cues such as peptide hormones and dietary nutrients. These signaling cues maintain the balance between self-renewal and differentiation of its resident stem cells. The mechanistic Target of Rapamycin Complex 1 (mTORC1) is implicated to play an important role in regulating this balance, although its downstream effectors in stem cells have been elusive. We have recently shown that the PASK protein kinase phosphorylates Wdr5 to stimulate muscle stem cell differentiation by epigenetically activating the Myogenin promoter. Here, we show that the PASK-Wdr5 signaling pathway is a nutrient-sensitive downstream target of mTORC1 in muscle stem cells. We show that phosphorylation of PASK, and in turn of Wdr5, by mTORC1 is required for the activation of Myogenin transcription, exit from the self-renewal and induction of the myogenesis program. Thus, mTOR connects the diverse extrinsic signaling cues to a central epigenetic process to regulate the muscle stem cell fate between self-renewal and differentiation.

Per-ARNT-Sim (PAS) domains are versatile sensors of intra- and extra-cellular metabolic environment. We have recently shown that in response to various pro-differentiation signaling cues, PAS domain containing protein Kinase (PAS Kinase, or PASK), regulates the differentiation of stem cells via phosphorylation of Wdr5. Wdr5 is a member of histone trimethylation at Lysine 4 (H3K4me3) enzymatic complexes. Wdr5 phosphorylation by PASK regulates H3K4me3 modification at the Myogenin promoter resulting in its activation during myogenesis. However, it remained unclear how PASK function is regulated in stem cells by differentiation signaling cues. Here we show that PASK is activated by nutrients such as glucose and amino acids and hormones such as insulin and IGF-1 in muscle stem cells. This activation of PASK is mediated by mTOR Complex 1 (mTORC1). PASK forms a nutrient-sensitive complex with mTORC1, and in response to nutrient signaling, mTOR directly phosphorylates PASK on multiple residues. This activation of PASK by mTOR recruits Wdr5 onto PASK, resulting in an increased Wdr5 phosphorylation. Finally, during myogenesis, both PASK and mTOR regulates the induction of the Myogenin mRNA expression and myogenesis program via Wdr5 phosphorylation. Since muscle is a metabolically active tissue, our findings suggest that metabolic cues such as glucose and amino-acids and insulin co-ordinates cellular metabolism with myogenesis program via mTOR-PASK signaling. Epigenetic control of Myogenesis program via mTOR-PASK-Wdr5 signaling. Metabolic cues such as insulin and nutrients activates PASK via mTOR mediated phosphorylation. Activated PASK phosphorylates Wdr5 to promote H3K4me3 modification on Myogenin promoter resulting in its activation. Myogenin regulates PASK expression in positive feedback manner to reinforce the differentiation program. Epigenetic control of Myogenesis program via mTOR-PASK-Wdr5 signaling. Metabolic cues such as insulin and nutrients activates PASK via mTOR mediated phosphorylation. Activated PASK phosphorylates Wdr5 to promote H3K4me3 modification on Myogenin promoter resulting in its activation. Myogenin regulates PASK expression in positive feedback manner to reinforce the differentiation program.

PAS domain containing protein kinase (Pask) is an evolutionarily conserved protein kinase implicated in energy homeostasis and metabolic regulation across eukaryotic species. We now describe an unexpected role of Pask in promoting the differentiation of myogenic progenitor cells, embryonic stem cells and adipogenic progenitor cells. This function of Pask is dependent upon its ability to phosphorylate Wdr5, a member of several protein complexes including those that catalyze histone H3 Lysine 4 trimethylation (H3K4me3) during transcriptional activation. Our findings suggest that, during myoblast differentiation, Pask stimulates the conversion of repressive H3K4me1 to activating H3K4me3 marks on the promoter of the differentiation gene myogenin (Myog) via Wdr5 phosphorylation. This enhances accessibility of the MyoD transcription factor and enables transcriptional activation of the Myog promoter to initiate muscle differentiation. Thus, as an upstream kinase of Wdr5, Pask integrates signaling cues with the transcriptional network to regulate the differentiation of progenitor cells.

Elevated hepatic synthesis of fatty acids and triglycerides, driven by hyperactivation of the SREBP-1c transcription factor, has been implicated as a causal feature of metabolic syndrome. SREBP-1c activation requires the proteolytic maturation of the endoplasmic-reticulum-bound precursor to the active, nuclear transcription factor, which is stimulated by feeding and insulin signaling. Here, we show that feeding and insulin stimulate the hepatic expression of PASK. We also demonstrate, using genetic and pharmacological approaches, that PASK is required for the proteolytic maturation of SREBP-1c in cultured cells and in the mouse and rat liver. Inhibition of PASK improves lipid and glucose metabolism in dietary animal models of obesity and dyslipidemia. Administration of a PASK inhibitor decreases hepatic expression of lipogenic SREBP-1c target genes, decreases serum triglycerides, and partially reverses insulin resistance. While the signaling network that controls SREBP-1c activation is complex, we propose that PASK is an important component with therapeutic potential.

PAS kinase (PASK) is a serine/threonine kinase implicated in the regulation of insulin gene expression via modulation of the transcription factor pancreas-duodenum homeobox-1 (PDX-1). However, the precise mechanisms by which PASK regulate PDX-1 expression and activity are unknown. The aim of this study was to test the hypothesis that PASK phosphorylates and thereby inactivates glycogen synthase kinase-3 beta (GSK3β), which is expected to alleviate PDX-1 protein degradation. In beta cells, GSK3β is known to phosphorylate PDX-1 on Ser residues at low glucose and trigger its proteasomal degradation. First, in vitro, we observed that PASK directly phosphorylates GSK3βon Ser9 (a known inactivating phosphorylation site of GSK3β). Second, we examined the effects of PASK on GSK3β and PDX-1 phosphorylation in isolated rat islets and HIT-T15 insulin-secreting lines by adenoviral transduction or transient transfection, respectively, of a wild-type (WT) or kinase-dead (KD, acting as a dominant-negative) form of PASK. We observed that overexpression of KD-PASK completely blocks the ability of glucose to phosphorylate GSK3β on Ser9. Third, WT-PASK mimics the effect of glucose in inhibiting PDX-1 serine phosphorylation, as assessed by immunoprecipitation. Fourth, overexpression of WT-PASK or KD-GSK3β protects PDX-1 protein from degradation in the presence of cycloheximide. We conclude that in beta cells, PASK phosphorylates and inactivates GSK3β, resulting in a decrease in PDX-1 serine phosphorylation and stabilization of PDX-1 protein.

Background: The enzyme PASK regulates the expression of PDX-1 and insulin in pancreatic β-cells via unknown mechanisms. Results: PASK enhances PDX-1 protein stability via phosphorylation of GSK3β on Ser9. Conclusion: PASK regulates insulin gene expression at least in part through inactivation of GSK3β and stabilization of PDX-1 protein. Significance: We identified GSK3β as a novel target of PASK in the regulation of pancreatic β-cell function. In pancreatic β-cells, glucose induces the binding of the transcription factor pancreatic duodenal homeobox-1 (PDX-1) to the insulin gene promoter to activate insulin gene transcription. At low glucose levels, glycogen synthase kinase 3β (GSK3β) is known to phosphorylate PDX-1 on C-terminal serine residues, which triggers PDX-1 proteasomal degradation. We previously showed that the serine/threonine Per-Arnt-Sim domain-containing kinase (PASK) regulates insulin gene transcription via PDX-1. However, the mechanisms underlying this regulation are unknown. In this study, we aimed to identify the role of PASK in the regulation of PDX-1 phosphorylation, protein expression, and stability in insulin-secreting cells and isolated rodent islets of Langerhans. We observed that glucose induces a decrease in overall PDX-1 serine phosphorylation and that overexpression of WT PASK mimics this effect. In vitro, PASK directly phosphorylates GSK3β on its inactivating phosphorylation site Ser9. Overexpression of a kinase-dead (KD), dominant negative version of PASK blocks glucose-induced Ser9 phosphorylation of GSK3β. Accordingly, GSK3β Ser9 phosphorylation is reduced in islets from pask-null mice. Overexpression of WT PASK or KD GSK3β protects PDX-1 from degradation and results in increased PDX-1 protein abundance. Conversely, overexpression of KD PASK blocks glucose-induction of PDX-1 protein. We conclude that PASK phosphorylates and inactivates GSK3β, thereby preventing PDX-1 serine phosphorylation and alleviating GSK3β-mediated PDX-1 protein degradation in pancreatic β-cells.

Enhanced activation of phosphoinositide 3-kinase (PI3K) is a hallmark of many human tumors because it promotes cell proliferation and survival through several mechanisms. One of these mechanisms is the phosphorylation of the serine and threonine kinase Akt at the cytosolic side of the plasma membrane by phosphoinositide-dependent protein kinase 1 (PDK1), which is recruited and activated by binding to the phosphoinositides produced by PI3K. We previously demonstrated increased nuclear accumulation of PDK1 in cells with enhanced PI3K activity. We report that nuclear PDK1 promoted cell proliferation by suppressing FOXO3A-dependent transcription of the gene encoding p27Kip1 (an inhibitor of cell cycle progression), whereas it enhanced cell survival by inhibiting the activation of c-Jun amino-terminal kinase. Cells with nuclear-localized PDK1 showed anchorage-independent growth, and when injected into mice, these cells induced the formation of solid tumors. In human prostate tumors, cytoplasmic localization of PDK1 correlated only with early-stage, low-risk tumors, whereas nuclear PDK1 localization correlated with high-risk tumors. Together, our findings suggest a role for nuclear-translocated PDK1 in oncogenic cellular transformation and tumor progression in mice and humans.

PAS domain containing serine/threonine kinase (PASK) is an evolutionary conserved protein kinase implicated in insulin resistance and type II diabetes. Specifically, we have shown a role for role for PASK in regulation hepatic lipogenesis pathways and insulin resistance using PASK knockout mice. It, however, remains unknown how PASK is regulated in cells and its downstream effectors. Here, we demonstrate that PASK is a nutrient responsive kinase. Serum and glucose stimulated the activity of PASK which was significantly suppressed by either rapamycin treatment or mTOR silencing by RNAi. In addition, the in vivo phosphorylation of PASK increases in response to serum and insulin treatment in an mTOR catalytic function dependent manner. Mutation of the phosphorylation sites to alanine suppresses serum stimulated phosphorylation and activation of PASK. Finally, we show that PASK physically associated with mTOR complex 1 in serum dependent manner, suggesting a direct role for mTOR complex 1 in regulating PASK function. Taken together, our findings provide the first evidence for the role of mTOR in regulating PASK activity and function in response to nutritional cues.